Transplantation of hemopoietic stem cells (hsc) in utero is likely to become an increasingly important form of therapy for congenital hemopoietic and metabolic disorders. Candidate diseases for replacement therapy include severe combined immunodeficiency, thalassemia and metabolic storage diseases. In spite of remarkable achievements in establishing allogeneic and xenogeneic chimeras by hsc introduction in utero, variable success in achieving chimerism and low levels of donor engraftment post-natally limit clinical application of prenatal hsc therapy. Our goal in this proposal is to determine the transplant conditions which allow prenatal cellular replacement in the murine hemopoietic system. We propose to apply sophisticated molecular and cellular techniques to define the role of hsc source and number, gestational age and recipient microenvironment in influencing donor hsc proliferation in utero. Increased understanding of the regulation of donor hsc proliferation in utero will allow definition of variables which modulate donor cell engraftment, and thereby facilitate design of cellular replacement strategies to effect disease cure. Specifically, we will do the following: 1) Define the relationship between hsc frequency and source in the donor inoculum and postnatal engraftment by introducing varying numbers of genetically-marked purified hsc in utero. 2) Define whether homing and proliferation of hsc transplanted in utero is determined by ontological maturity of the fetal microenvironment or by the changing developmental potential of hsc. 3) Determine whether allogeneic hsc are at a proliferative disadvantage compared to their syngeneic counterparts and whether exogenous exposure to developmentally-regulated cytokines (i.e., c-kit ligand, IL-3 and IL-6) promotes their proliferation in utero. 4) Determine whether lineage- specific hemopoietic defects facilitate donor hsc proliferation in utero and determine the engraftment level required for cure of genetically- mutant mice with hemopoietic disorders and lysosomal storage disease. Additionally, we will evaluate, in collaboration with other investigators, whether stem cells expanded in culture support hemopoiesis when introduced prenatally. It is anticipated that data generated in these studies will lead to new insights about strategies to improve success of prenatal hsc transplantation.